Butanol is an intermediate compound with a very wide range of uses, such as in cosmetics, perfumes, hormones, hygiene products, industrial coating agents, paint additives, fibers, plastic monomers, medical supplies, vitamins, antibiotics, agricultural chemicals, and the like, and is very useful (Dune, Biotechnol J, 2:1525-1534, 2007).
Up to the 1980s, methods for producing butanol, acetone and ethanol by fermenting sugars with Clostridium strains were utilized as typical methods for preparing butanol (Weizmann, U.S. Pat. No. 1,315,585). Since the 1980s, an oxo process for synthesizing butanol from propylene originated from petroleum has been widely used. However, the method for preparing butanol based on petroleum has drawbacks in that the process is complicated due to the use of high temperature and high pressure, and a large amount of hazardous waste and carbon dioxide are released therefrom (Tsuchida et al., Ind. Eng. Chem. Res., 45:8634, 2006). Recently, the demand for eco-friendly production of butanol from renewable sources through microbial fermentation has greatly increased.
However, in order to produce butanol at industrially applicable levels using microorganisms, it is essential that the production method should have good butanol selectivity, yield and productivity, namely butanol production amount per unit hour. However, among wild type and recombinant microorganisms for producing bio-butanol, a microorganism satisfying all these conditions has yet to be found.
Specifically, a wild type Clostridium acetobutylicum Strain ATCC824 is known to produce acetone, ethanol and butanol in a mass ratio of about 3:1:6 through fermentation and a small amount of acetic acid and butyric acid, wherein the yield of the wild type strain is about 25%, and the final concentration is about 10 g/L or so. Like Clostridium acetobutylicum, microorganisms having an acetyl-CoA biosynthetic pathway and a butyryl-CoA biosynthetic pathway are generally known to synthesize acetone, butanol and ethanol in the pathways as shown in FIG. 1. Recently, with the development of metabolic engineering, endeavors to produce butanol more effectively have continued. Especially, since the genomic sequence of Clostridium acetobutylicum is recently disclosed, research relating to metabolic pathway manipulation has been actively performed.
For example, co-overexpression of adhE1 and ctfAB genes in Clostridium acetobutylicum M5 in which a megaplasmid having butanol production related genes (adc, ctfAB and adhE1 (alcohol/aldehyde dehydrogenase) and adhE2 (alcohol/aldehyde dehydrogenase)) was deleted showed that butanol selectivity was enhanced to a mass ratio of 0.78, but the co-overexpression had limits in that productivity and yield were greatly reduced as growth of the strain was inhibited and the production of acetic acid increased (Lee, et al., Biotechnology Journal, 4:1432-1440, 2009; Lee, et al., WO 2009/082148).
In the case where pta converting acetyl-CoA into acetate is deleted, and in the case where both pta and buk converting butyryl-CoA into butyrate are deleted and aad (alcohol/aldehyde dehydrogenase) is overexpressed, both cases are reported to enhance butanol concentration, selectivity and yield. However, both cases still have limits in view of productivity and stability of strains (Lee et al., WO 2011/037415). Furthermore, in the case where CtfB coding CoA transferase (CoAT) was further deleted from a mutant strain from which pta and buk are deleted, the strain still shows low productivity (LEE et al., WO 2011/037415).
In addition, in the case where a mutant Clostridium beijerinckii BA101 strain derived by random mutation is subjected to fermentation utilizing maltodextrin as a carbon source, it is reported that 18.6 g/l, of butanol is produced (Ezeji et al., Appl. Microbiol. Biotechnol., 63:653, 2004). However, even if the recombinant strain is employed, the strain has no industrial applicability due to low production of final product, i.e. butanol.
In addition, reports say that the concentration of acetone is decreased and butanol selectivity is increased by deleting ctfAB encoding CoA transferase or adc (acetoacetate decarboxylase). However, this report has problems in view of strain stability and the final concentration of butanol less than 10 g/L (Jiang et al., Metab. Eng., 11(4-5):284-291, 2009).
The present inventors made research to find microorganisms having good butanol selectivity, yield and productivity. As a result, the present inventors found that a recombinant mutant microorganism capable of producing butanol with high yield, high selectivity and high productivity can be manufactured by deleting pta and buk simultaneously which are butyrate and acetate production related genes, and co-overexpressing CtfAB which encodes CoA transferase (CoAT) and adhE (alcohol/aldehyde dehydrogenase) which converts butyryl-CoA to butanol. Based on this finding, the present invention has been completed.